CN111620949A - Antibodies that bind human LAG-3, methods of making, and uses thereof - Google Patents

Abstract

The invention discloses an antibody or an antigen binding fragment thereof capable of specifically binding human LAG-3, which has a brand-new epitope bound with human LAG-3 and good biological activity for enhancing mixed lymphocyte reaction, can be applied to preparation of a medicament for cancer immunotherapy, and has good clinical application prospect.

Description

Antibodies that bind human LAG-3, methods of making, and uses thereof

Technical Field

The invention relates to the field of antibodies, and more particularly discloses an antibody binding to human LAG-3, a preparation method and a use thereof.

Background

Various types of malignancies have now become the major killers of humans and the incidence is rising year by year. Although the conventional operations, chemotherapy and radiotherapy have certain curative effect, the traditional Chinese medicine composition usually brings great damage and toxic and side effects to the body of a patient. Although the existing targeted monoclonal antibody medicines (such as trastuzumab targeting HER 2) and targeted small molecule kinase inhibitors (such as imatinib targeting certain tyrosine kinases) have favorable clinical effects in tumor treatment, the medicines are only effective on tumors expressing specific targets, the types of cancers which can be treated are limited, the objective response rate is low, and the tumors applying the antibodies are easy to generate drug resistance so as to relapse and progress again. The tumor immunotherapy applies immunological principle and method to activate immune system, break immune tolerance state of immune system to tumor, enhance the recognition of immune cell to tumor antigen, and stimulate and enhance anti-tumor immune response of organism, so that the immune system of organism can inhibit and kill tumor cell, and finally the goal of treating tumor can be achieved. Tumor immunotherapy has recently received much attention and is the focus of the tumor therapy field. In recent years, significant information on tumor immunotherapy has emerged. At present, programmed death receptor 1 (PD-1) is undoubtedly the most dazzling target in cancer immunotherapy, and monoclonal anti-tumor drugs such as nivolumab and pembrolizumab, which target it, have demonstrated strong anti-tumor activity in clinical treatment of some tumor types such as melanoma, non-small cell lung cancer, etc., and have been approved for clinical application by FDA in the united states. Because of its excellent curative effect and innovativeness, immunotherapy for tumors is judged by the journal of science in 2013 as the most important scientific breakthrough every year. The tumor immunotherapy is expected to become a revolution in the field of tumor therapy after operations, chemotherapy, radiotherapy and targeted therapy.

T cells play an important role in the immune system, but activation of T cells relies on the antigen presenting cells to digest and re-present foreign harmful antigens to an antigen form that the T cells can recognize. T cells and antigen presenting cells have a population of proteins involved in regulation, which function to help regulate T cell receptor signaling, and these helper receptors are divided into 2 major classes, one class being costimulatory receptors responsible for transmitting activating signals, and the other class being co-inhibitory receptors transmitting inhibitory signals, and these inhibitory molecules are now referred to as immune checkpoint receptors and ligands. The star molecule PD-1 mentioned above is a typical receptor of immune checkpoints and belongs to the CD28 superfamily members, and its important ligand is programmed death ligand-1 (PD-L1). The binding of PD-1 to PD-L1 mediates a co-inhibitory signal of T cell activation, negatively regulating T cell activation and proliferation. In addition, chinese scientists have demonstrated that PD-L1 is highly expressed in tumor tissue and modulates the function of tumor-infiltrating CD8+ T cells. Currently, immunomodulatory therapies targeting PD-1/PD-L1 have met with great success in clinical antitumor therapy.

Lymphocyte activation gene-3 (LAG-3) is mainly expressed in activated T lymphocytes and is an immune negative regulation molecule similar to PD-1, and the main ligand of LAG-3 is a MHC II molecule family, which can compete with CD4 molecule to bind MHC II ligand with higher affinity and transduce inhibitory signals to the inside of the cell, thereby negatively regulating the activation, proliferation and dynamic balance of the cell, and the molecular mechanism is very similar to PD-1. LAG-3 can help T cells to maintain an immune tolerance state or enable continuously activated T cells to be in a depletion state, has the functions of maintaining homeostasis and participating in immune negative regulation, and is closely related to the occurrence and development of tumors. At present, some preliminary studies show that the antibody for blocking LAG-3 can enhance the immune response, and can play a remarkable synergistic activation role when being combined with the PD-1 antibody, so that the potential in the field of cancer immunotherapy is huge.

At present, most of the research and development of monoclonal antibody medicines taking LAG-3 as a target point internationally are still in an early stage, main research and development companies are international medicine headaches such as Bristol-Myers Squibb (BMS) and Novartis (Novartis), wherein the anti-LAG-3 monoclonal antibody BMS-986016 of the Baishimei Guibao is in a first clinical stage, the main research targets are the safety and effectiveness of combined use with PD-1 antibody nivolumab, and research results show that BMS-986016 and nivolumab show good curative effect in clinical treatment of melanoma. In addition, anti-LAG-3 antibodies can also be used to treat autoimmune diseases, the basic mechanism is to kill activated T cells through antibody-dependent cell-mediated cytotoxicity (ADCC), thereby achieving the purpose of suppressing immune response and treating autoimmune diseases. However, there is still a need to develop more novel, specific and efficient LAG-3 targeted drugs for clinical application in immunotherapy.

Disclosure of Invention

In order to solve the technical problems, the inventor of the invention carries out a large number of experiments, from antigen immunization, hybridoma screening, antibody expression purification to biological activity identification, obtains two antibodies of murine 134 and 2-34 (namely 5E7) which specifically bind to human LAG-3 through screening, and further constructs and obtains Chimeric antibodies 134-Chimeric, 5E7-Chimeric and humanized antibodies 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 on the basis of the antibodies. The present study shows that murine antibodies 134 and 2-34 (i.e., 5E7) have a novel epitope for binding to human LAG-3. Cell level experimental results show that humanized antibodies 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 can effectively block the binding of LAG-3 to Raji cells and enhance the secretion of IL-2 by SEA stimulated PBMCs. Accordingly, the antibodies or antigen-binding fragments thereof that bind human LAG-3 developed in the present invention can be used in the preparation of a medicament for cancer immunotherapy.

Accordingly, it is a first object of the present invention to provide an antibody or antigen-binding fragment thereof that binds human LAG-3.

It is a second object of the invention to provide another antibody or antigen-binding fragment thereof that binds human LAG-3.

It is a third object of the present invention to provide a nucleotide sequence encoding said antibody or antigen-binding fragment thereof that binds human LAG-3.

The fourth purpose of the invention is to provide an expression vector containing the nucleotide sequence.

The fifth object of the present invention is to provide a host cell containing the expression vector.

The sixth object of the present invention is to provide a method for producing the antibody or antigen-binding fragment thereof that binds human LAG-3.

The seventh object of the present invention is to provide a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds human LAG-3.

An eighth object of the present invention is to provide the use of the antibody or antigen-binding fragment thereof that binds human LAG-3 or the pharmaceutical composition.

In order to achieve the purpose, the invention adopts the following technical scheme:

a first aspect of the invention provides an antibody or antigen-binding fragment thereof that binds human LAG-3, which binds to an epitope of human LAG-3 comprising the amino acid sequence: AAAPGHPLA (SEQ ID NO: 50).

According to a preferred embodiment of the invention, the human LAG-3 epitope to which it binds comprises the amino acid sequence: GPPAAAPGHPLA (SEQ ID NO: 48) or AAAPGHPLAPGPHPAAPSS (SEQ ID NO: 49).

A second aspect of the invention provides an antibody or antigen-binding fragment thereof that binds human LAG-3, comprising:

(a) heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 11 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 13, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 14 or SEQ ID NO: 43 is shown; or

(b) Heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 15, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 16, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 17 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 18, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 19, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: shown at 20.

According to the invention, the antibody is a monoclonal antibody or a polyclonal antibody. Preferably, the antibody is a monoclonal antibody.

According to the present invention, the antibody is a murine antibody, a chimeric antibody, a humanized antibody or the like. Preferably, the antibody is a humanized antibody.

According to the present invention, the antigen binding fragment includes a Fab fragment, a F (ab') 2 fragment, a Fv fragment, and the like.

According to a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

(a) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 4 is shown in the specification;

(b) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 6, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 8 is shown in the specification;

(c) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 22, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 24; and

(d) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 26, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 28.

According to a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 heavy chain constant regions and a light chain constant region selected from the group consisting of kappa or lambda light chain constant regions. Preferably, the heavy chain constant region is an IgG4 heavy chain constant region and the light chain constant region is a kappa light chain constant region. More preferably, the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID NO: 30, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO: shown at 34.

According to a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain and a light chain selected from the group consisting of:

(a) the amino acid sequence of the heavy chain is shown as SEQ ID NO: 32, and the amino acid sequence of the light chain is shown as SEQ ID NO: 36 is shown; and

(b) the amino acid sequence of the heavy chain is shown as SEQ ID NO: 38, and the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 40.

A third aspect of the invention provides a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that binds human LAG-3 as described in any one of the above.

According to a preferred embodiment of the invention, the nucleotide sequence comprises:

(a) as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 3, a nucleotide sequence encoding a light chain variable region;

(b) as shown in SEQ ID NO: 5, as shown in SEQ ID NO: 7, a nucleotide sequence encoding a light chain variable region;

(c) as shown in SEQ ID NO: 21, as shown in SEQ ID NO: 23, a nucleotide sequence encoding a light chain variable region; or

(d) As shown in SEQ ID NO: 25, as shown in SEQ ID NO: 27, and a nucleotide sequence encoding a light chain variable region.

According to a preferred embodiment of the invention, the nucleotide sequence comprises the nucleotide sequence as set forth in SEQ ID NO: 29, and the nucleotide sequence encoding the heavy chain constant region as set forth in SEQ ID NO: 33, and a nucleotide sequence encoding a light chain constant region.

According to a preferred embodiment of the invention, the nucleotide sequence comprises:

(a) as shown in SEQ ID NO: 31, as shown in SEQ ID NO: 35, a nucleotide sequence encoding a light chain; or

(b) As shown in SEQ ID NO: 37, as shown in SEQ ID NO: 39 encoding a light chain.

In a fourth aspect, the present invention provides an expression vector comprising a nucleotide sequence as defined in any one of the above.

In a fifth aspect, the present invention provides a host cell comprising an expression vector as described above.

A sixth aspect of the invention provides a method of making an antibody or antigen-binding fragment thereof that binds human LAG-3, as described above, comprising the steps of:

(a) culturing a host cell as described above under expression conditions such that the antibody or antigen-binding fragment thereof that binds human LAG-3 is expressed;

(b) isolating and purifying the antibody or antigen-binding fragment thereof that binds human LAG-3 of (a).

A seventh aspect of the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds human LAG-3 as described in any one of the above, and a pharmaceutically acceptable carrier.

According to a preferred embodiment of the invention, the pharmaceutical composition further comprises a PD-1 inhibitor. Preferably, the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that binds to PD-1.

An eighth aspect of the invention provides the use of an antibody or antigen-binding fragment thereof that binds human LAG-3, as defined in any one of the above, or a pharmaceutical composition as defined in any one of the above, in the manufacture of a medicament for the treatment of cancer.

According to the invention, the cancer is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, classical Hodgkin lymphoma, urothelial cancer, colorectal cancer, liver cancer and the like.

Has the advantages that:

the invention discloses an antibody or an antigen binding fragment thereof capable of specifically binding human LAG-3, which has a brand-new epitope bound with human LAG-3 and good biological activity for enhancing mixed lymphocyte reaction, can be applied to preparation of a medicament for cancer immunotherapy, and has good clinical application prospect.

Drawings

FIG. 1 shows the results of the effect of murine anti-human LAG-3 antibody on mixed lymphocyte reaction, wherein FIG. 1A shows the first round of screening and FIG. 1B shows the second round of screening.

FIG. 2 is a result of comparison of relative affinities between a humanized antibody and a chimeric antibody against human LAG-3 antibody.

FIG. 3 is a graph showing the effect of the humanized anti-human LAG-3 antibody on the enhancement of IL-2 secretion by PBMC from different donors in FIGS. 3A and 3B, respectively.

FIG. 4 shows the results of the binding effect of humanized anti-human LAG-3 antibody to cell surface LAG-3.

FIG. 5 is a schematic representation of epitopes of anti-human LAG-3 antibody.

FIG. 6 shows the blocking effect of humanized anti-human LAG-3 antibody on the binding of LAG-3 to Raji cells.

Detailed Description

In the present invention, the term "LAG-3" refers to lymphocyte activation gene 3, also referred to as CD 223.

In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)", which are heterotetrameric glycan proteins of about 150000 daltons having the same structural features, are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a constant region consisting of three domains, CH1, CH2, and CH 3. Each light chain has a variable region (VL) at one end and a constant region at the other end, the light chain constant region comprising a domain CL; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. The constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC), and the like. Heavy chain constant regions include IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; light chain constant regions include κ (Kappa) or λ (Lambda).

The antibody of the present invention includes a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., bispecific antibody) formed from at least two antibodies, and the like.

In the present invention, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

In the present invention, the term "antigen-binding fragment" refers to a fragment of an antibody that is capable of specifically binding to an antigen (e.g., human LAG-3). Examples of the antigen-binding fragment of the present invention include Fab fragment, F (ab') 2 fragment, Fv fragment, and the like. Fab fragments are fragments generated by papain digestion of antibodies. The F (ab') 2 fragment is a fragment produced by digestion of an antibody with pepsin. Fv fragments are composed of dimers in which the heavy and light chain variable regions of an antibody are in close, non-covalent association.

In the present invention, the term "Fc fragment" means that papain can cleave an antibody into two identical Fab fragments and one Fc fragment, i.e., a crystallizable fragment (Fc), which is composed of the CH2 and CH3 domains of the antibody. The Fc region has no antigen binding activity and is the site of antibody interaction with effector molecules or cells.

In the present invention, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and the light chain variable region. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)).

The antibody of the present invention includes a murine antibody, a chimeric antibody, a humanized antibody and the like.

In the present invention, the term "murine antibody" refers to an antibody derived from a rat or a mouse, preferably a mouse. The murine antibody of the invention is obtained by immunizing a mouse by using the extracellular domain of human LAG-3 as an antigen and screening hybridoma cells. Preferably, the murine antibodies of the invention are antibodies 134 and 2-34 (i.e., 5E 7).

In the present invention, the term "chimeric antibody" refers to an antibody comprising heavy chain variable region and light chain variable region sequences derived from one species and constant region sequences derived from another species, for example, an antibody having murine heavy chain variable region and light chain variable region linked to human constant regions. Preferably, the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region and light chain variable region sequences of murine antibody Nos. 134 and 2-34 (i.e., 5E7), respectively, to human constant regions. More preferably, the heavy chain of the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region sequences of murine antibodies 134 and 2-34 (i.e., 5E7) to the human IgG4 heavy chain constant region, respectively, and the light chain is obtained by splicing the light chain variable region sequences of murine antibodies 134 and 2-34 (i.e., 5E7) to the human Kappa light chain constant region, respectively. Most preferably, Chimeric antibodies of the invention include 134-Chimeric and 5E 7-Chimeric.

In the present invention, the term "humanized antibody" means an antibody in which the CDRs are derived from a non-human species (preferably a mouse), and the remaining part of the antibody molecule (including the framework region and the constant region) is derived from a human antibody. In addition, framework region residues may be altered to maintain binding affinity. Preferably, the humanized antibody of the present invention is obtained by recombining the CDR regions of murine antibody Nos. 134 and 2-34 (i.e., 5E7) and the non-CDR regions derived from a human antibody and back-mutating the buried residues, residues that directly interact with the CDR regions, and residues that significantly affect the conformation of VL and VH of antibody Nos. 134 and 2-34 (i.e., 5E 7). More preferably, the humanized antibodies of the invention include 134-Hu-IgG4-C91S and 5E7-Hu-IgG 4.

In the present invention, the terms "epitope" and "human LAG-3 epitope" refer to a region on human LAG-3 to which an antibody specifically binds. Preferably, the epitope of human LAG-3 of the present invention is located within the extracellular domain of human LAG-3, said extracellular domain of human LAG-3 comprising the amino acid sequence as set forth in SEQ ID NO: 41. More preferably, the human LAG-3 epitope of the invention comprises the amino acid sequence: AAAPGHPLA (SEQ ID NO: 50). Most preferably, the human LAG-3 epitope of the invention comprises the amino acid sequence: GPPAAAPGHPLA (SEQ ID NO: 48) or AAAPGHPLAPGPHPAAPSS (SEQ ID NO: 49).

In the present invention, the term "antibody that binds human LAG-3" or "anti-human LAG-3 antibody" refers to an antibody that specifically binds to an epitope of human LAG-3 the term "specifically binds" refers to an antibody that binds less than 1 × 10^-7M or less, preferably 1 × 10^-8M or less, more preferably 1 × 10^-9M or less affinity (KD) binding.

In the present invention, the term "expression vector" may be pTT5, pSECtag series, pCGS3 series, pCDNA series vectors, etc., and other vectors for use in mammalian expression systems, etc., the expression vector including a fusion DNA sequence to which appropriate transcription and translation regulatory sequences are ligated.

In the present invention, the term "host cell" refers to a cell suitable for expressing the above expression vector, and may be a eukaryotic cell, such as mammalian or insect host cell culture system, which can be used for the expression of the fusion protein of the present invention, and CHO (Chinese Hamster Ovary), HEK293, COS, BHK and derived cells of the above cell can be suitable for the present invention.

In the present invention, the term "pharmaceutical composition" means that the antibody or antigen-binding fragment thereof binding to human LAG-3 of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation composition that can ensure the conformational integrity of the amino acid core sequence of the antibody or antigen-binding fragment thereof binding to human LAG-3 disclosed herein, while also protecting the multiple functional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation) to exert therapeutic effects more stably.

The following examples and experimental examples are intended to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2 interpretation, Cold spring harbor Laboratory Press.

Example 1 preparation of human LAG-3 protein and anti-LAG-3 Positive control antibody

Human LAG-3 extracellular domain amino acid sequence fromhttps://www.uniprot.org/uniprot/P18627And the amino acid sequence is shown as SEQ ID NO: 41, codon optimization and gene synthesis were performed by Biotechnology engineering (Shanghai) Co., Ltd, and the synthesized gene fragment was cloned into pUC57 vector. Designing primers, and amplifying a coding region (SEQ ID NO: 41) of an Extracellular Domain (ECD) of LAG-3 and a coding region (SEQ ID NO: 51) of an Fc region of human immunoglobulin IgG1 by using a PCR (polymerase chain reaction) method; recovering the amplified fragments, splicing the fragments together by recombinant PCR, introducing a signal peptide (MGVKVLFALICIAVAEA, SEQ ID NO: 42) coding region, and introducing corresponding enzyme cutting sites at two ends; the pTT5 expression vector (purchased from NRC Biotechnology research Institute) and the aforementioned recombinant PCR fragment were cleaved with restriction enzymes; purifying the two groups of enzyme digestion products, connecting the two groups of enzyme digestion products by using ligase, transforming the enzyme digestion products into escherichia coli TOP10 competent cells, and coating the escherichia coli TOP10 competent cells in an LB (Amp resistance) plate culture medium for overnight culture; selecting a monoclonal colony, extracting plasmids after culture and amplification, and cutting the plasmids by using restriction endonuclease to identify whether a target gene fragment is inserted; selecting qualified positive clones for sequencing; after the clone with the completely correct sequence is selected and amplified, the plasmid is extracted. The LAG-3 expression vector constructed above was transferred to HEK293E cells (purchased from NRCBIOtechnology Research Institute) by Polyethyleneimine (PEI) for expression, after 5 days, cell supernatants were collected and purified by Protein A affinity chromatography, and the obtained Protein was named LAG-3-ECD-hFc and used for immunization of mice and subsequent screening process. HEK293E cells were cultured in FreeStyle 293Expression Medium (purchased from Thermo Fisher scientific) chemically defined serum-free Medium. Protein quantification was performed by uv spectrophotometry.

The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-LAG-3 monoclonal antibody as a positive control were derived from SEQ ID NO: 12 and 14, carrying out codon optimization and synthesizing genes by the company Limited in Biotechnology engineering (Shanghai), respectively connecting the heavy chain variable region gene and the light chain variable region gene with a human IgG4 heavy chain constant region (the nucleotide sequence is shown as SEQ ID NO: 29, and the amino acid sequence is shown as SEQ ID NO: 30) and a human Kappa light chain constant region (the nucleotide sequence is shown as SEQ ID NO: 33, and the amino acid sequence is shown as SEQ ID NO: 34), introducing a signal peptide sequence (SEQ ID NO: 42) to construct complete heavy chain and light chain genes, and respectively cloning the complete heavy chain and light chain genes into a pTT5 expression vector according to the similar method. The pTT5 expression vectors containing heavy chain and light chain genes are combined by the same method and transferred into HEK293E cells for expression, after 5 days, cell supernatants are collected, and the antibody is purified by Protein A affinity chromatography, and the obtained antibody is named as Anti-LAG 3.5. HEK293E cells were cultured in FreeStyle 293Expression Medium (purchased from Thermo Fisher Scientific) chemically defined serum-free Medium. Protein quantification was determined by uv spectrophotometry.

Example 2 immunization of mice with human LAG-3-ECD-hFc as antigen and preparation and screening of hybridomas

Diluting the LAG-3-ECD-hFc prepared in example 1 with normal saline to a suitable concentration, mixing with equal volume of Freund's complete adjuvant (purchased from Sigma), and performing multi-point subcutaneous injection on 4-5 week-old Balb/c mice (purchased from Shanghai Ling biological technology Limited, animal production license number: SCXK 2013-; three weeks later, the LAG-3-ECD-hFc was diluted with physiological saline and mixed with an equal volume of Freund's incomplete adjuvant (purchased from Sigma), and the mice were injected subcutaneously with multiple spots after completion of the phacoemulsification; this step was repeated again two weeks later; all mice were bled a small amount of blood at the seventh day after the third immunization to isolate serum, and the serum titer was determined by conventional ELISA; mice with serum antibody titers of >10000 were selected and seven days later, each mouse was injected with 10. mu.g of antigen protein via tail vein.

On the third day after intravenous injection of antigen protein, a certain number of mice were selected, sacrificed and dissected to take out spleens, ground, collected splenic cells andand counting for preparing the hybridoma. Myeloma Sp2/0 cells (from the cell bank of the culture Collection of the Chinese academy of sciences) were selected as cell fusion partners. Sp2/0 cells in cell culture incubator (37 ℃, 5% CO)₂) The medium used in the medium culture is RPMI-1640 complete medium (prepared by adding 10% fetal bovine serum and 1% penillin-Streptomycin to RPMI-1640 basic medium, which are all purchased from ThermoFisher Scientific), the medium is replaced one day before cell fusion to make the cells in a better growth state, the hybridoma cells are prepared by electrofusion, the hybridoma screening process comprises mixing two cells according to the ratio of spleen cells to Sp2/0 cells to 2: 1, centrifuging to remove supernatant, washing the cells three times with cell fusion buffer (purchased from BTX), and precipitating the cells 1 × 10⁷Suspension in cell fusion buffer at a density of/ml; adding 2ml cell suspension into cell fusion pool, placing on electrofusion apparatus ECM2001 (purchased from BTX), and processing under certain conditions (AC60V, 30S; DC 1700V, 40. mu.S, 3X; POSTAC 60V, 3S); then gently transferring the treated cells to a pre-heated RPMI-1640 complete medium at 37 ℃ and standing at room temperature for 1 hour; the treated cells are pressed 10 times⁴Per well density was inoculated into 96-well plates at 100. mu.l/well, 100. mu.l/well of complete medium containing 2 × HAT (50 × HAT available from Thermo Fisher Scientific, used as appropriate diluted with medium as needed) was added to each well the next day, the old medium was replaced with fresh RPMI-1640 complete medium containing 1 × HAT on the fourth and seventh days, respectively, and samples were taken on the ninth day after fusion for ELISA testing to identify positive clones.

Detection of positive clones (clones capable of secreting anti-LAG-3 antibody) was performed by ELISA method: coating the ELISA plate by using LAG-3-ECD-hFc antigen, wherein the coating amount is 100 ng/hole; incubation at room temperature for 2 hours followed by washing with PBST (PBS containing 0.05% Tween-20); add 200. mu.l PBST solution containing 1% Bovine Serum Albumin (Bovine Serum Albumin, BSA) to each well for blocking, incubate for 1 hour at room temperature; after washing with PBST, draining, and storing in a refrigerator for later use. When in detection, 100 mu l/hole of hybridoma culture supernatant is added into an enzyme label plate, and the incubation is carried out for about 1 hour; with PBSWashing for 3 times; HRP-Goat-Anti-Mouse IgG antibody (purchased from KPL) was diluted appropriately with 1% BSA in PBST and incubated at room temperature for about 1 hour; PBST washing 3 times; adding 100 μ l of color developing solution (with TMB as substrate) into each well for color development; 2M H was then added per well₂SO₄Stopping the reaction by using a stop solution; immediately measuring the OD value of each well at a wavelength of 450nm by using a microplate reader (Molecular Device); the data was analyzed to determine positive clones.

Subcloning was performed by limiting dilution method by preparing hybridoma cell suspension of positive clone, adjusting cell density to dilution of 10 or 20 cells per ml with HT medium (HT medium prepared by diluting 100 × HT solution with RPMI-1640 complete medium to desired concentration, 100 × HT from Thermo Fisher Scientific), inoculating 96-well plate at each dilution with 0.2ml per well and average number of hybridoma cells per well of 2 or 4, placing in CO₂Culturing in a cell culture box for 7-10 days, observing under an inverted microscope when macroscopic clones appear, marking holes in which only a single clone grows, and taking supernatant for ELISA detection (detection method is described above); taking the hole with ELISA detection positive to perform amplification culture in a 24-hole plate, continuing to perform a new round of subcloning by using a limiting dilution method, selecting not less than 24 cloning holes, performing ELISA detection, and obtaining a monoclonal hybridoma cell strain for stably expressing a target antibody when the detection shows 100% positive; and (3) carrying out amplification culture on the monoclonal hybridoma cell strain, and carrying out frozen preservation.

The stable monoclonal cell line was cultured in an expanded state for about 5 to 7 days in serum-free medium HybriGRO SF (purchased from Corning), and after removing the precipitate from the cell culture supernatant by centrifugation and filtration, the murine antibody was purified using Protein G (purchased from GEHealthcare). Through several times of fusion and screening, more than 200 stable hybridoma monoclonal cell strains are successfully established, and corresponding murine antibodies are obtained through purification and used for further experiments.

Example 3 Effect of murine anti-human LAG-3 antibodies on Mixed lymphocyte responses

Here, the activity of murine Anti-human LAG-3 antibody was evaluated using Mixed Lymphocyte Reaction (MLR) and Anti-LAG3.5 was added as a positive control.

The MLR experimental method is described below: peripheral Blood Mononuclear Cells (PBMC) were isolated from human Blood using Histopaque (purchased from Sigma), subpopulations of monocytes in PBMC were isolated by the adherence method, and then monocyte differentiation into induced dendritic cells was induced using IL-4 in combination with GM-CSF (p38MAPK-inhibited dendritic cells induced and vitamin regulation T-Cell-mediated immune suppression. Natcommu. 2014Jun 24; 5:4229.doi: 10.1038/ncoms 5229.); seven days later, digesting and collecting the induced dendritic cells; PBMCs were isolated from blood from another donor using the method described above, and CD4 positive T cells were isolated therefrom using a MACS magnet and CD4MicroBeads (available from Miltenyi Biotec); dendritic cells to be induced (10)⁴One/well) and isolated CD4 positive T cells (10)⁵Per well) as 10: 1, and then inoculating the mixture into a 96-well plate, wherein each well is 150 mu l; after 2 hours, 50. mu.l of murine anti-LAG-3 antibody diluted with AIM-V medium was added to the above 96-well plate; setting a positive control antibody Anti-LAG3.5 and an unrelated isotype control antibody which does not bind to LAG-3; place 96-well plates in a cell incubator (37 ℃, 5% CO)₂) Incubating for 3-4 days, taking a proper amount of culture supernatant, and detecting the secretion of IL-2 by using double-antibody sandwich ELISA (related detection reagents are purchased from BD Biosciences); OD450 was read with a multifunctional microplate reader (Molecular Device), and data analysis and mapping were performed with GraphPad Prism 6. In this example, cells were cultured in AIM-V (purchased from Thermo Fisher Scientific) serum-free medium.

The experimental results are shown in FIGS. 1A and 1B, and two rounds of screening (FIGS. 1A and 1B) were performed to obtain two murine anti-human LAG-3 antibodies, monoclonal antibodies No. 134 and No. 2-34 (i.e., 5E7), which significantly enhance IL-2 secretion by MLR.

Example 4 determination of nucleotide and amino acid sequences of anti-human LAG-3 antibodies, preferably of murine origin

According to the screening results of example 3, monoclonal antibodies No. 134 and No. 5E7 were finally picked as lead antibodies. Total RNA was extracted from hybridoma monoclonal cell lines corresponding to 134 and 5E7 using TRIzol (purchased from Thermo Fisher Scientific), mRNA was reverse-transcribed into cDNA using a reverse transcription kit (purchased from Takara), the light chain variable region and the heavy chain variable region genes of murine-derived LAG anti-human-3 Antibody were amplified by PCR using a primer set (Antibody Engineering, Volume 1, Edded by Roland Kontermann and Stefan Dubel; the sequence of the primer set is from page 323) reported in the literature, and then the PCR products were cloned into pMD18-T vector, sequenced and the variable region gene sequences were analyzed.

The sequence information for clone number 134 is as follows: the heavy chain variable region gene sequence has a full length of 360bp, 120 amino acid residues are coded, and the nucleotide sequence is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2 is shown in the specification; the light chain variable region gene sequence has the full length of 321bp, encodes 107 amino acid residues, and has the nucleotide sequence shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4, respectively.

Amino acid sequence of heavy chain variable region cloned No. 134

EVQLQQSGPVLVKPGASVKMSCKASGYTLTAYYMNWVKQSRGKSLEWIGVINPYNGDSSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARDDGYYRWYFDVWGTGTTVTVSS(SEQ ID NO：2)

Amino acid sequence of light chain variable region cloned No. 134

DIQMTQSPSSLSASLGERVSLTCRASQDIGSRLNWLQQGPDGSIKRLIYATSSLESGVPKRFSGSRSGSDYFLTISSLESEDFVDYYCLQCGSSPPTFGGGTKLEIK(SEQ ID NO：4)

The sequence information for clone No. 5E7 is as follows: the heavy chain variable region gene sequence has a total length of 348bp, codes 116 amino acid residues, and has a nucleotide sequence shown as SEQ ID NO: 5, and the amino acid sequence is shown as SEQ ID NO: 6 is shown in the specification; the light chain variable region gene sequence has the full length of 321bp, encodes 107 amino acid residues, and has the nucleotide sequence shown as SEQ ID NO: 7, and the amino acid sequence is shown as SEQ ID NO: shown in fig. 8.

Amino acid sequence of heavy chain variable region cloned at No. 5E7

EVKLVESGGGLVKPGGSLKLSCAASGFTFSDDYMAWFRQTPEKRLEWVASISHGGDYIYYADNLKGRFTISRDNAKNTLYLQMSSLKSEDTAIYFCSRDRRSIDYWGQGTTLTVSS(SEQ ID NO：6)

Amino acid sequence of light chain variable region cloned in No. 5E7

DIQMTQITSSLSASLGDRVTITCRASQDISNYLSWYQQKPDGTIKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISDLEQEDIATYFCQQGKTLPYTFGGGTKLERK(SEQ ID NO：8)

Example 5 humanization of murine anti-human LAG-3 antibodies

The amino acid sequences of the heavy chain variable region and the light chain variable region in example 4 were analyzed to determine 3 antigen Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs) of heavy and light chains of antibodies nos. 134 and 5E7, respectively, according to the Kabat encoding rules.

Wherein, the amino acid sequence of the heavy chain complementarity determining region cloned No. 134 is HCDR 1: AYYMN (SEQ ID NO: 9), HCDR 2: VINPYNGDSSYNQKFKG (SEQ ID NO: 10) and HCDR 3: DDGYYRWYFDV (SEQ ID NO: 11), and the amino acid sequence of the light chain complementarity determining region is LCDR 1: RASQDIGSRLN (SEQ ID NO: 12), LCDR 2: ATSSLES (SEQ ID NO: 13) and LCDR 3: LQCGSSPPT (SEQ ID NO: 14).

Wherein, the amino acid sequence of the heavy chain complementarity determining region of clone No. 5E7 is HCDR 1: DDYMA (SEQ ID NO: 15), HCDR 2: SISHGGDYIYYADNLKG (SEQ ID NO: 16) and HCDR 3: DRRSIDY (SEQ ID NO: 17), and the amino acid sequence of the light chain complementarity determining region is LCDR 1: RASQDISNYLS (SEQ ID NO: 18), LCDR 2: YTSRLHS (SEQ ID NO: 19) and LCDR 3: QQGKTLPYT (SEQ ID NO: 20).

In thathttps://www.ncbi.nlm.nih.gov/igblast/In the method, the amino acid sequence of the heavy chain variable region of the antibody No. 134 is subjected to homology comparison with the sequence of a human immunoglobulin variable region embryonic system (Germline), IGHV1-46 x 01 with the highest homology with No. 134 is selected as a heavy chain variable region humanized template, and the corresponding CDRs in IGHV1-46 x 01 are replaced by murine heavy chain CDRs to construct a heavy chain CDR-grafted variable region. Similarly, light chain CDR-grafted variable regions were constructed by selecting the IGKV1-39 x 01 germline sequence as the light chain variable region humanised template and replacing the corresponding CDRs in IGKV1-39 x 01 with murine 134 light chain CDRs, by homology comparison. Amino acid sites within some framework regions are then back-mutated based on the heavy and light chain CDR grafted variable regions. The reversion is to shift the CDRsCertain amino acids (amino acids important for maintaining antibody structure and affinity) within the framework regions of the plant variable regions are mutated to amino acids at corresponding positions in the murine framework regions. In making back mutations, the amino acid sequence is encoded according to Kabat rules, with the position of each amino acid indicated by Kabat encoding. Preferably, the heavy chain CDR grafted variable region has 44-site G back mutation to S, 48-site M mutation to I, 67-site V mutation to A, 69-site M mutation to L, 71-site R mutation to V, 73-site T mutation to K, and 78-site V mutation to A. Preferably, the light chain CDR grafted variable region 36Y is back mutated to L, 43A to S, 44P to I, 46L to R, 66G to R, 69T to S and 71F to Y. It is noted that the light chain CDR3(SEQ ID NO: 14) of antibody 134 contains an extra cysteine (Kabat encoded position 91, C91) which may cause structural abnormalities in the antibody, and is mutated to a serine herein (C91S, SEQ ID NO: 43). The amino acid sequences were added to the ends of the heavy and light chain CDR-grafted variable regions carrying the above back mutations: WGQGTKVEIK (SEQ ID NO: 44) and FGQGTKVEIK (SEQ ID NO: 45), to construct a complete humanized heavy and light chain variable region, and the relevant gene sequences were codon optimized and gene synthesized by Biotechnology engineering (Shanghai) GmbH.

The finally obtained 134 humanized heavy chain variable region (134-Hu-VH) gene sequence has the full length of 354bp, codes 118 amino acid residues, and has a nucleotide sequence shown as SEQ ID NO: 21, and the amino acid sequence is shown as SEQ ID NO: 22; the gene sequence of the 134 humanized light chain variable region (134-Hu-VL-C91S) has the full length of 321bp, codes 107 amino acid residues, and has a nucleotide sequence shown as SEQ ID NO: 23, and the amino acid sequence is shown as SEQ ID NO: as shown at 24.

Amino acid sequence of 134 humanized heavy chain variable region (134-Hu-VH)

QVQLVQSGAEVKKPGASVKVSCKASGYTLTAYYMNWVRQAPGQSLEWIGVINPYNGDSSYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDDGYYRWYFDVWGQGTLVTVSS (SEQ ID NO: 22, where the underlined part is the heavy chain complementarity determining region)

Amino acid sequence of 134 humanized light chain variable region (134-Hu-VL-C91S)

DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKSIKRLIYATSSLESGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQSGSSPPTFGQGTKVEIK (SEQ ID NO: 24, where the underlined part is the light chain complementarity determining region)

The antibody No. 5E7 was humanized in the same manner. The most homologous IGHV3-21 x 01 with 5E7 is selected as the humanized template of heavy chain variable region, and the corresponding CDR in IGHV3-21 x 01 is replaced by murine heavy chain CDR 5E7 to construct heavy chain CDR grafted variable region. Similarly, by homology comparison, IGKV1-33 × 01 germline sequences were selected as light chain variable region humanized templates, and mouse-derived No. 5E7 light chain CDRs were substituted for the corresponding CDRs in IGKV1-33 × 01 to construct light chain CDR-grafted variable regions. Amino acid sites within some framework regions are then back-mutated based on the heavy and light chain CDR grafted variable regions. In making back mutations, the amino acid sequence is encoded according to Kabat rules, with the position of each amino acid indicated by Kabat encoding. Preferably, the V at position 37 of the heavy chain CDR grafted variable region is back mutated to F, the G at position 44 is mutated to R, the S at position 49 is mutated to A, the Y at position 91 is mutated to F, and the A at position 93 is mutated to S. Preferably, the light chain CDR grafted variable region 43A mutation to T, 44P mutation to I, 71F mutation to Y, 87Y mutation to F mutation. The amino acid sequences were added to the ends of the heavy and light chain CDR-grafted variable regions carrying the above back mutations: WGQGTKVEIK (SEQ ID NO: 44) and FGQGTKVEIK (SEQ ID NO: 45), to construct a complete humanized heavy and light chain variable region, and the relevant gene sequences were codon optimized and gene synthesized by Biotechnology engineering (Shanghai) GmbH.

The finally obtained 5E7 humanized heavy chain variable region (5E7-Hu-VH) gene sequence has the full length of 354bp, codes 118 amino acid residues, and has a nucleotide sequence shown as SEQ ID NO: 25, and the amino acid sequence is shown as SEQ ID NO: 26 is shown; the full length of the 5E7 humanized light chain variable region (5E7-Hu-VL) gene sequence is 321bp, 107 amino acid residues are coded, and the nucleotide sequence is shown as SEQ ID NO: 27, and the amino acid sequence is shown as SEQ ID NO: shown at 28.

Amino acid sequence of humanized heavy chain variable region of 5E7 (5E7-Hu-VH)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDDYMAWFRQAPGKRLEWVASISHGGDYIYYADNLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCSRDRRSIDYWGQGTLVTVSS (SEQ ID NO: 26, where the underlined part is the heavy chain complementarity determining region)

Amino acid sequence of 5E7 humanized light chain variable region (5E7-Hu-VL)

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKTIKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTLPYTFGQGTKVEIK (SEQ ID NO: 28, where the underlined part is the light chain complementarity determining region)

The artificially synthesized 134 humanized heavy chain variable region is connected with a constant region (the nucleotide sequence is shown as SEQ ID NO: 29, and the amino acid sequence is shown as SEQ ID NO: 30) of human immunoglobulin IgG4 to construct a complete 134 humanized heavy chain which is named as 134-Hu-IgG4-HC (the nucleotide sequence is shown as SEQ ID NO: 31, and the amino acid sequence is shown as SEQ ID NO: 32); the 134 humanized light chain variable region was linked to the human immunoglobulin Kappa chain constant region (nucleotide sequence shown in SEQ ID NO: 33, amino acid sequence shown in SEQ ID NO: 34) to construct a complete 134 humanized light chain, designated 134-Hu-LC-C91S (nucleotide sequence shown in SEQ ID NO: 35, amino acid sequence shown in SEQ ID NO: 36).

Amino acid sequence of human immunoglobulin IgG4 constant region

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：30)

Amino acid sequence of 134 humanized heavy chain 134-Hu-IgG4-HC

QVQLVQSGAEVKKPGASVKVSCKASGYTLTAYYMNWVRQAPGQSLEWIGVINPYNGDSSYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDDGYYRWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 32, where the underlined part is the heavy chain complementarity determining region)

Amino acid sequence of human immunoglobulin Kappa chain constant region

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：34)

Amino acid sequence of 134 humanized light chain 134-Hu-LC-C91S

DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKSIKRLIYATSSLESGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQSGSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 36, wherein the underlined part is the light chain complementarity determining region)

The artificially synthesized 5E7 humanized heavy chain variable region is connected with a human immunoglobulin IgG4 constant region (the nucleotide sequence is shown as SEQ ID NO: 29, and the amino acid sequence is shown as SEQ ID NO: 30) to construct a complete 5E7 humanized heavy chain which is named as 5E7-Hu-IgG4-HC (the nucleotide sequence is shown as SEQ ID NO: 37, and the amino acid sequence is shown as SEQ ID NO: 38); the 5E7 humanized light chain variable region was linked to the human immunoglobulin Kappa chain constant region (nucleotide sequence shown in SEQ ID NO: 33, amino acid sequence shown in SEQ ID NO: 34) to construct a complete 5E7 humanized light chain, named 5E7-Hu-LC (nucleotide sequence shown in SEQ ID NO: 39, amino acid sequence shown in SEQ ID NO: 40).

Amino acid sequence of 5E7 humanized heavy chain 5E7-Hu-IgG4-HC

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDDYMAWFRQAPGKRLEWVASISHGGDYIYYADNLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCSRDRRSIDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 38, wherein the underlined part is the heavy chain complementarity determining region)

5E7 humanized light chain 5E7-Hu-LC amino acid sequence

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKTIKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 40, wherein the underlined part is the light chain complementarity determining region)

The 134-Hu-IgG4-HC gene and the 134-Hu-LC-C91S gene are respectively constructed into a pTT5 expression vector, and the constructed expression vectors are co-transfected into HEK293E cells by PEI for transient expression. HEK293E cells were cultured in FreeStyle 293Expression Medium (purchased from Thermo Fisher Scientific) serum-free Medium, centrifuged 5 days later and the cell supernatant was collected, and the antibody was purified by Protein A affinity chromatography, and finally the prepared antibody was named 134-Hu-IgG 4-C91S. The expression vectors for the humanized heavy and light chains of 5E7 were constructed in the same manner, and the antibody was expressed and purified in the same manner, and finally prepared and named 5E7-Hu-IgG 4.

In addition, the heavy chain variable region and the light chain variable region of the murine antibody 134 were respectively spliced with the human immunoglobulin IgG4 heavy chain constant region (nucleotide sequence shown in SEQ ID NO: 29, amino acid sequence shown in SEQ ID NO: 30) and Kappa light chain constant region (nucleotide sequence shown in SEQ ID NO: 33, amino acid sequence shown in SEQ ID NO: 34) by recombinant PCR method, and respectively constructed into pTT5 expression vector, the antibody was expressed and purified by the above method, and the prepared Chimeric antibody was named 134-Chimeric. A Chimeric antibody of the 5E7 antibody, 5E7-Chimeric, was obtained in the same manner.

The effect of humanization on the affinity between antibody and antigen was examined by ELISA. The enzyme-labeled plate (10 ng/well) was coated with LAG-3-ECD-His (His-tagged LAG-3 extracellular segment, available from Nano Biological Inc.), incubated at room temperature for 2 hours, and then PBST (containing 0.05% Tween-20 PBS) wash; blocking was performed by adding 200. mu.l of blocking solution (PBST solution containing 1% BSA) to each well; washing the ELISA plate by PBST and then patting the plate dry; adding an anti-human LAG-3 antibody which is diluted in a sealing solution in a gradient manner into an ELISA plate, incubating for about 1 hour at room temperature, and then washing the ELISA plate; adding a Goat-Anti-Human IgG (Fc specific) secondary antibody (purchased from Sigma) diluted by a blocking solution, incubating at room temperature for about 1 hour, and washing an enzyme label plate; adding 100 μ l of color developing solution (TMB substrate) into each well for color development; then 50. mu.l of 2M H was added to each well₂SO₄Terminating the color development reaction; OD values of each well were measured at a wavelength of 450nm using SpectraMax 190(Molecular Device); data analysis and mapping were performed using GraphPad Prism6, and EC50 was calculated.

The experimental results are shown in FIG. 2, and Anti-LAG3.5, 134-Chimeric, 134-Hu-IgG4-C91S, 5E7-Chimeric and 5E7-Hu-IgG4 can effectively bind to LAG-3, and EC50 is 27.63ng/ml, 8.685ng/ml, 8.782ng/ml, 9.741ng/ml and 9.078ng/ml respectively, and the corresponding humanized antibody is basically consistent with EC50 of the Chimeric antibody, which indicates that the affinity among the antibody antigens is not reduced by the humanized modification. Wherein the isotype control is an IgG4 antibody that does not bind LAG-3.

Example 6 examination of the enhancement of IL-2 secretion by PBMCs of humanized anti-human LAG-3 antibodies

Staphylococcal Enterotoxin (SEA) is a superantigen capable of cross-linking MHC class II molecules of Antigen Presenting Cells (APCs) and T Cell Receptors (TCRs) on the surface of T cells, thereby activating a large number of T Cell clones and strongly stimulating an immune response. LAG-3 is expressed after T cell activation, and the LAG-3 can be combined with MHC II molecules on the surface of APC to play an immunosuppressive role. In this example, the functional activity of humanized anti-human LAG-3 antibody was evaluated by detecting IL-2 secretion after PBMC stimulation with SEA and addition of anti-human LAG-3 antibody.

The experimental methods are described below: cloning SEA gene with histidine tag into pET28a vector, expressing the protein with colibacillus system, and purifying SEA with nickel affinity chromatographic column; PBMCs were isolated from human blood using Histopaque, washed with PBS and centrifuged; using RPMI-1640 medium (containing 10% fetal bovine serum)Serum), adding the above-mentioned self-made SEA (SEQ ID NO: 52) to a final concentration of 1ng/ml, and then inoculating PBMC into round-bottomed 96-well cell culture plates, each well 2 × 10⁵Cells, each volume of 150. mu.l; then, 50. mu.l of anti-human LAG-3 antibody was added to each well in a gradient, and the 96-well plate was placed in a cell incubator (37 ℃ C., 5% CO)₂) Incubating for 2 days; taking a proper amount of culture supernatant, and detecting the secretion amount of IL-2 by using double-antibody sandwich ELISA (related detection reagents are purchased from BD Biosciences); OD450 was read with a microplate reader (Molecular Device), and data analysis and mapping were performed with GraphPad Prism 6.

The PBMCs in figures 3A and 3B were from different donors, respectively. The experimental results show that Anti-LAG3.5, 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 can effectively enhance IL-2 secretion of SEA-stimulated PBMCs. EC50 of Anti-LAG3.5, 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 in the experiment shown in FIG. 3A were 98.09ng/ml, 50.71ng/ml and 78.12ng/ml, respectively; in the experiment shown in FIG. 3B, the three antibodies had EC50 of 16.21ng/ml, 10.85ng/ml and 3.068ng/ml, respectively. The results of two experiments show that the functional activity of stimulating activated PBMC to secrete IL-2 by 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 is stronger than that of Anti-LAG 3.5. Wherein the isotype control is an IgG4 antibody that does not bind LAG-3.

Example 7 binding of humanized anti-human LAG-3 antibodies to cell surface LAG-3

Superantigen SEA was able to activate T cell clones in PBMC, which activated T cells expressed LAG-3. In this example, the binding effect of the humanized anti-human LAG-3 antibody of the present invention on cell surface LAG-3 was measured by flow cytometry.

The experimental procedure is described as follows, PBMC are isolated from human blood with Histopaque, washed 2 times with PBS, harvested by centrifugation, resuspended in RPMI-1640 medium (containing 10% fetal bovine serum), SEA is added to a final concentration of 1ng/ml, and PBMC are then seeded into round bottom 96 well cell culture plates, 2 × 10 per well⁵(ii) individual cells; place 96-well plates in a cell incubator (37 ℃, 5% CO)₂) Incubating for 2 days; washing cells in a 96-well plate by using PBS, centrifuging and then discarding supernatant; biotinylation of the antibody with Biotin N-hydroxyysticimide ester (cat/cat: H1759-100MG, available from Sigma) was carried outAs per the manufacturer's instructions; adding a biotin-labeled anti-human LAG-3 antibody which is diluted in a gradient manner, incubating for about 1 hour, and then washing the cells for 2 times by using PBS; PE-Streptavidin (purchased from BD Biosciences) appropriately diluted with PBS containing 1% BSA was added to the above cells, incubated for about 1 hour, and then the cells were washed 2 times with PBS; adding 4% paraformaldehyde to fix cells; the cells were examined for PE fluorescence intensity on a flow Cytometer (CytoFLEX Cytometer System, available from beckmann coulter); processing the experimental data with flow cytometer analysis software and calculating the average fluorescence intensity; data analysis and mapping were performed using GraphPad Prism6 and EC50 was calculated.

The experimental results are shown in FIG. 4, and Anti-LAG3.5, 134-Hu-IgG4-C91S and 5E7-Hu-IgG4 can effectively bind to LAG-3 on the cell surface, EC50 is 71.68ng/ml, 80.45ng/ml and 53.13ng/ml respectively, and the binding capacities of the three are basically consistent. Wherein isotype control was IgG4 antibody that did not bind LAG-3, and the ordinate was mean fluorescence intensity.

Example 8 epitope analysis of anti-human LAG-3 antibody

The first immunoglobulin-like domain of the extracellular domain of LAG-3 contains an exposed "extra loop" having the amino acid sequence: GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 46). The peptide fragment has been reported in the literature to play an important role in the binding of LAG-3 to MHC class II molecules (Baixeras E, Huard B, Miossec C, et. characters of the lymphocyte activation gene 3-encoded protein. A newalign and for human leukocyte activation class II antigens) [ J]Journal of Experimental Medicine,1992,176(2): 327-. To examine the binding of the anti-human LAG-3 antibody of the present invention to the peptide and to determine the binding epitope of each antibody, the following peptide scanning experiment was performed: chemically synthesizing a series of partially overlapped peptide fragments which cover the whole 'extra loop' region, and connecting a biotin label to the N end of the peptide fragments; coating an enzyme label plate (200 ng/hole) with Streptavidin, washing the enzyme label plate, and then blocking with a blocking solution (PBST solution containing 1% BSA); the biotinylated peptide fragment was diluted to 1. mu.g/ml with blocking solution, added to an ELISA plate, incubated at room temperature for 1 hour to allow it to be captured by Streptavidin, and thenWashing the enzyme label plate; adding an anti-human LAG-3 antibody which is diluted by a confining liquid in a gradient manner, incubating for about 1 hour at room temperature, and then washing an ELISA plate; the binding of Anti-Human LAG-3 antibody to the peptide fragment was detected by adding a Goat-Anti-Human IgG (Fc specific) secondary antibody (purchased from Sigma) diluted appropriately in blocking solution, and incubated at room temperature for about 1 hour; the color was developed by adding 100. mu.l of a developing solution (TMB substrate) to each well, followed by adding 50. mu.l of 2M H to each well₂SO₄Stopping the reaction by using a stop solution; OD values of each well were measured at a wavelength of 450nm using SpectraMax 190(Molecular Devices, Inc.); data analysis and mapping were performed using GraphPad Prism6, and EC50 was calculated. The results of the peptide fragment scanning experiments are summarized in table 1.

TABLE 1 summary of the results of the peptide fragment scanning experiments

Remarking: "-" indicates no significant binding, "+" indicates significant binding, and more "+" indicates stronger binding; the smaller the EC50 the more "+".

If an antibody binds to multiple peptides clearly, the shortest peptide that overlaps is the epitope of the antibody. Table 1 experimental results show that the epitope of Anti-LAG3.5 antibody is PHPAAPSSW (SEQ ID NO: 47), and the epitope of antibody 134 is GPPAAAPGHPLA (SEQ ID NO: 48); whereas the epitope of antibody No. 5E7 was AAAPGHPLAPGPHPAAPSS (SEQ ID NO: 49). The correlation results are shown in fig. 5.

Example 9 blocking of LAG-3 binding to Raji cells by humanized anti-human LAG-3 antibody

Raji cells are Burkitt lymphoma cells, derived from B lymphocytes, that highly express MHC-class II molecules. This example uses flow cytometry to determine the blocking effect of the humanized anti-human LAG-3 antibodies of the present invention on the binding of LAG-3 to Raji cell surface MHC-class II molecules.

The experimental procedure was as follows: raji cells (purchased from American Type Culture Collection, abbreviated ATCC;

CCL-86^TM) Washing machineWashed 2 times and then seeded into round bottom 96 well cell culture plates, 2 × 10 per well⁵Centrifuging individual cells, and discarding supernatant; biotinylated LAG-3 (purchased from ACROBIOSystems) was diluted to 0.5. mu.g/ml with 1% BSA in PBS, and then anti-human LAG-3 antibody was further diluted in this solution in a gradient, and then the prepared mixed solution containing biotinylated LAG-3 and anti-human LAG-3 antibody was added to the above cells, incubated at room temperature for about 1 hour, and the cells were washed with PBS for 2 times; PE-Streptavidin (purchased from BDbiosciences) was appropriately diluted with PBS containing 1% BSA, added to the above cells, incubated for about 1 hour, and the cells were washed 2 times with PBS; adding 4% paraformaldehyde to fix cells; the cells were examined for PE fluorescence intensity on a flow cytometer (cytoflex cytometry System, available from Beckman Coulter); processing the experimental data with flow cytometer analysis software and calculating the average fluorescence intensity; data analysis and mapping were performed using GraphPad Prism6 and EC50 was calculated.

As shown in FIG. 6, Anti-LAG3.5 and 134-Hu-IgG4-C91S both effectively block the binding of LAG-3 to Raji cells, and IC50 is 256.9ng/ml and 288.8ng/ml respectively, and the blocking abilities of the two are basically consistent. The effect of 5E7-Hu-IgG4 on LAG-3 binding to Raji cells is different from Anti-LAG3.5 and 134-Hu-IgG 4-C91S: at high concentrations, 5E7-Hu-IgG4 only partially blocked the binding of LAG-3 to Raji cells; at low concentrations, 5E7-Hu-IgG4 instead promoted the binding of LAG-3 to Raji cells. Wherein isotype control was IgG4 antibody that did not bind human LAG-3, and negative control indicated background fluorescence of Raji cells in the absence of LAG-3.

Sequence listing

<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH

<120> antibody binding to human LAG-3, preparation method and use thereof

<130>2019

<160>52

<170>SIPOSequenceListing 1.0

<210>1

<211>360

<212>DNA

<213>Mus musculus

<400>1

gaggtccagc tgcaacagtc tggacctgtg ctggtgaagc ctggggcttc agtaaagatg 60

tcctgtaagg cttctggata cacactcact gcctactata tgaactgggt gaagcaaagt 120

cgtggaaaga gccttgagtg gattggagtt attaatcctt acaacggtga ttctagctac 180

aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagcctac 240

atggagctca acagcctgac atctgaggac tctgcagtct attactgtgc aagggatgat 300

ggttactacc gatggtactt cgatgtctgg ggcacaggga ccacggtcac cgtctcctca 360

<210>2

<211>120

<212>PRT

<213>Mus musculus

<400>2

Glu Val Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ala Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ser Arg Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Tyr Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Gly Tyr Tyr Arg Trp Tyr Phe Asp Val Trp Gly Thr

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>3

<211>321

<212>DNA

<213>Mus musculus

<400>3

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggacattggt agtagattaa actggcttca gcagggacca 120

gatggaagta ttaaacgcct gatctacgcc acatccagtt tagaatctgg tgtccccaaa 180

aggttcagtg gcagtaggtc tgggtcagat tactttctca ccatcagcag ccttgagtct 240

gaagattttg tagactatta ctgtctacag tgtggtagtt ctcctccgac gttcggtgga 300

ggcaccaagctggaaatcaa a 321

<210>4

<211>107

<212>PRT

<213>Mus musculus

<400>4

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg

20 25 30

Leu Asn Trp Leu Gln Gln Gly Pro Asp Gly Ser Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Glu Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Phe Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Cys Gly Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>5

<211>348

<212>DNA

<213>Mus musculus

<400>5

gaagtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt gacgattaca tggcttggtt tcgccagact 120

cctgagaaaa ggctggagtg ggtcgcatcc attagtcatg gtggtgatta catctactat 180

gcagacaatt tgaaggggcg attcaccatc tccagagaca atgccaagaa caccctgtac 240

ctgcaaatga gcagtctgaa gtctgaggac acagccatat atttctgttc aagagatcgg 300

cgatccattg actactgggg ccaaggcacc actctcacag tctcctca 348

<210>6

<211>116

<212>PRT

<213>Mus musculus

<400>6

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asp

20 25 30

Tyr Met Ala Trp Phe Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser His Gly Gly Asp Tyr Ile Tyr Tyr Ala Asp Asn Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Phe Cys

85 90 95

Ser Arg Asp Arg Arg Ser Ile Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210>7

<211>321

<212>DNA

<213>Mus musculus

<400>7

gatatccaga tgacacagat tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcacttgca gggcaagtca ggacattagc aattatttaa gctggtatca gcagaaacca 120

gatggaacta ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcga cctggagcaa 240

gaagatatcg ccacttactt ttgccaacag ggtaaaacgc ttccgtacac gttcggaggg 300

gggaccaagc tggaaagaaa a 321

<210>8

<211>107

<212>PRT

<213>Mus musculus

<400>8

Asp Ile Gln Met Thr Gln Ile Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asp Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Arg Lys

100 105

<210>9

<211>5

<212>PRT

<213>Mus musculus

<400>9

Ala Tyr Tyr Met Asn

1 5

<210>10

<211>17

<212>PRT

<213>Mus musculus

<400>10

Val Ile Asn Pro Tyr Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210>11

<211>11

<212>PRT

<213>Mus musculus

<400>11

Asp Asp Gly Tyr Tyr Arg Trp Tyr Phe Asp Val

1 5 10

<210>12

<211>11

<212>PRT

<213>Mus musculus

<400>12

Arg Ala Ser Gln Asp Ile Gly Ser Arg Leu Asn

1 5 10

<210>13

<211>7

<212>PRT

<213>Mus musculus

<400>13

Ala Thr Ser Ser Leu Glu Ser

1 5

<210>14

<211>9

<212>PRT

<213>Mus musculus

<400>14

Leu Gln Cys Gly Ser Ser Pro Pro Thr

1 5

<210>15

<211>5

<212>PRT

<213>Mus musculus

<400>15

Asp Asp Tyr Met Ala

15

<210>16

<211>17

<212>PRT

<213>Mus musculus

<400>16

Ser Ile Ser His Gly Gly Asp Tyr Ile Tyr Tyr Ala Asp Asn Leu Lys

1 5 10 15

Gly

<210>17

<211>7

<212>PRT

<213>Mus musculus

<400>17

Asp Arg Arg Ser Ile Asp Tyr

1 5

<210>18

<211>11

<212>PRT

<213>Mus musculus

<400>18

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ser

1 5 10

<210>19

<211>7

<212>PRT

<213>Mus musculus

<400>19

Tyr Thr Ser Arg Leu His Ser

1 5

<210>20

<211>9

<212>PRT

<213>Mus musculus

<400>20

Gln Gln Gly Lys Thr Leu Pro Tyr Thr

1 5

<210>21

<211>360

<212>DNA

<213>Synthetic

<400>21

caagtgcagc tggtgcagtc cggcgccgaa gtgaagaagc ctggcgcctc cgtgaaggtg 60

agctgcaagg cttccggcta caccctgacc gcctactaca tgaactgggt gaggcaggcc 120

cctggccagt ccctggaatg gatcggcgtg atcaacccct acaacggcga ctcctcctat 180

aaccagaagt tcaagggccg ggccaccctg accgtggaca agtccaccag caccgcctac 240

atggagctgt cctccctgag gtccgaggac accgccgtct actactgcgc cagggacgac 300

ggctactaca ggtggtactt cgacgtgtgg ggccagggca ccctggtgac agtgtcctcc 360

<210>22

<211>120

<212>PRT

<213>Synthetic

<400>22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ala Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Tyr Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Gly Tyr Tyr Arg Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>23

<211>321

<212>DNA

<213>Synthetic

<400>23

gacatccaga tgacccagtc cccttcctcc ctgtccgctt ccgtgggcga cagggtgacc 60

atcacctgta gggcctccca ggacatcggc tccaggctga actggctgca gcagaagccc 120

ggcaagtcca tcaagcggct gatctacgcc acctcctccc tggagtccgg cgtgccttcc 180

aggttctccg gctccaggtc cggctccgac tacaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctacta ctgcctgcag tccggctcct cccctcctac cttcggccag 300

ggcaccaagg tggagatcaa g 321

<210>24

<211>107

<212>PRT

<213>Synthetic

<400>24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg

20 25 30

Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ser Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ser Gly Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>25

<211>348

<212>DNA

<213>Synthetic

<400>25

gaagtgcagc tggtggagtc cggaggcgga ctggtgaagc ctggaggctc cctgaggctg 60

tcctgtgccg cttccggctt caccttctcc gacgactaca tggcctggtt caggcaggcc 120

cctggaaaga ggctggagtg ggtggcttcc atctcccacg gcggcgacta catctactac 180

gccgacaacc tgaagggcag gttcaccatc tccagggaca acgccaagaa ctccctgtac 240

ctgcagatga actccctgag ggccgaggac accgccgtgt acttctgctc cagggacagg 300

aggtccatcg actattgggg ccagggcacc ctggtgacag tgtcctcc 348

<210>26

<211>116

<212>PRT

<213>Synthetic

<400>26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asp

20 25 30

Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser His Gly Gly Asp Tyr Ile Tyr Tyr Ala Asp Asn Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ser Arg Asp Arg Arg Ser Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>27

<211>321

<212>DNA

<213>Synthetic

<400>27

gacatccaga tgacccagtc cccttcctcc ctgtccgctt ccgtgggcga cagggtgacc 60

atcacctgca gggccagcca ggacatctcc aactacctgt cctggtacca gcagaagccc 120

ggcaagacca tcaagctgct gatctactac acctccaggc tgcacagcgg cgtgcctagc 180

aggttctccg gttctggctc cggcaccgac tacaccttca ccatcagctc cctgcagccc 240

gaggacatcg ccacctactt ctgccagcag ggcaagaccc tgccctacac cttcggccag 300

ggcaccaagg tggagatcaa g 321

<210>28

<211>107

<212>PRT

<213>Synthetic

<400>28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Thr Ile Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>29

<211>981

<212>DNA

<213>Homo

<400>29

gcaagtacca agggacctag tgttttccct cttgcacctt gctccaggtc aacatcagag 60

tccacagctg ctcttggatg tctcgttaag gactacttcc cagagccagt taccgtatcc 120

tggaactccg gagctttgac aagcggcgtt catacattcc cagctgtgtt gcagagttct 180

gggttgtaca gtttgagctc agtggtgacc gtgccttcat cttctttggg cactaagacc 240

tacacctgca acgtggatca caagccaagc aacaccaagg tggataagag ggtggagtcc 300

aagtacggcc caccatgtcc tccatgtcca gcccctgaat ttttgggcgg gccttctgtc 360

tttctgtttc ctcctaaacc taaagatacc ctgatgatca gccgcacacc cgaagtcact 420

tgtgtggtcg tggatgtgtc tcaggaagat cccgaagtgc agtttaactg gtatgtcgat 480

ggcgtggaag tgcataatgc caaaactaag ccccgcgaag aacagttcaa cagcacttat 540

cgggtcgtgt ctgtgctcac agtcctccat caggattggc tgaatgggaa agaatataag 600

tgcaaggtga gcaataaggg cctccccagc agcatcgaga agactattag caaagccaaa 660

gggcagccac gggaacccca ggtgtacact ctgcccccct ctcaggagga gatgactaaa 720

aatcaggtct ctctgacttg tctggtgaaa gggttttatc ccagcgacat tgccgtggag 780

tgggagtcta atggccagcc cgagaataat tataagacaa ctccccccgt cctggactct 840

gacggcagct ttttcctgta ttctcggctg acagtggaca aaagtcgctg gcaggagggc 900

aatgtcttta gttgcagtgt catgcatgag gccctgcaca atcactatac acagaaaagc 960

ctgtctctga gtctgggcaa a 981

<210>30

<211>327

<212>PRT

<213>Homo

<400>30

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>31

<211>1341

<212>DNA

<213>Synthetic

<400>31

caagtgcagc tggtgcagtc cggcgccgaa gtgaagaagc ctggcgcctc cgtgaaggtg 60

agctgcaagg cttccggcta caccctgacc gcctactaca tgaactgggt gaggcaggcc 120

cctggccagt ccctggaatg gatcggcgtg atcaacccct acaacggcga ctcctcctat 180

aaccagaagt tcaagggccg ggccaccctg accgtggaca agtccaccag caccgcctac 240

atggagctgt cctccctgag gtccgaggac accgccgtct actactgcgc cagggacgac 300

ggctactaca ggtggtactt cgacgtgtgg ggccagggca ccctggtgac agtgtcctcc 360

gcaagtacca agggacctag tgttttccct cttgcacctt gctccaggtc aacatcagag 420

tccacagctg ctcttggatg tctcgttaag gactacttcc cagagccagt taccgtatcc 480

tggaactccg gagctttgac aagcggcgtt catacattcc cagctgtgtt gcagagttct 540

gggttgtaca gtttgagctc agtggtgacc gtgccttcat cttctttggg cactaagacc 600

tacacctgca acgtggatca caagccaagc aacaccaagg tggataagag ggtggagtcc 660

aagtacggcc caccatgtcc tccatgtcca gcccctgaat ttttgggcgg gccttctgtc 720

tttctgtttc ctcctaaacc taaagatacc ctgatgatca gccgcacacc cgaagtcact 780

tgtgtggtcg tggatgtgtc tcaggaagat cccgaagtgc agtttaactg gtatgtcgat 840

ggcgtggaag tgcataatgc caaaactaag ccccgcgaag aacagttcaa cagcacttat 900

cgggtcgtgt ctgtgctcac agtcctccat caggattggc tgaatgggaa agaatataag 960

tgcaaggtga gcaataaggg cctccccagc agcatcgaga agactattag caaagccaaa 1020

gggcagccac gggaacccca ggtgtacact ctgcccccct ctcaggagga gatgactaaa 1080

aatcaggtct ctctgacttg tctggtgaaa gggttttatc ccagcgacat tgccgtggag 1140

tgggagtcta atggccagcc cgagaataat tataagacaa ctccccccgt cctggactct 1200

gacggcagct ttttcctgta ttctcggctg acagtggaca aaagtcgctg gcaggagggc 1260

aatgtcttta gttgcagtgt catgcatgag gccctgcaca atcactatacacagaaaagc 1320

ctgtctctga gtctgggcaa a 1341

<210>32

<211>447

<212>PRT

<213>Synthetic

<400>32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ala Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Tyr Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Gly Tyr Tyr Arg Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr LysPro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210>33

<211>321

<212>DNA

<213>Homo

<400>33

cgcactgtgg ctgcccccag tgttttcata tttcccccca gtgatgagca actgaagtcc 60

ggcacagcct ctgttgtatg tctgctgaat aatttttatc cacgggaggc caaggtgcag 120

tggaaggtgg acaatgccct gcagtctggg aactctcaag agagtgtgac agagcaggac 180

agtaaagaca gcacctatag cctcagcagc accctgaccc tgtctaaagc cgactatgaa 240

aaacacaaag tgtatgcctg cgaagtgacc catcaggggc tcagctctcc cgttaccaag 300

agctttaacc gaggcgaatg t 321

<210>34

<211>107

<212>PRT

<213>Homo

<400>34

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala AspTyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>35

<211>642

<212>DNA

<213>Synthetic

<400>35

gacatccaga tgacccagtc cccttcctcc ctgtccgctt ccgtgggcga cagggtgacc 60

atcacctgta gggcctccca ggacatcggc tccaggctga actggctgca gcagaagccc 120

ggcaagtcca tcaagcggct gatctacgcc acctcctccc tggagtccgg cgtgccttcc 180

aggttctccg gctccaggtc cggctccgac tacaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctacta ctgcctgcag tccggctcct cccctcctac cttcggccag 300

ggcaccaagg tggagatcaa gcgcactgtg gctgccccca gtgttttcat atttcccccc 360

agtgatgagc aactgaagtc cggcacagcc tctgttgtat gtctgctgaa taatttttat 420

ccacgggagg ccaaggtgca gtggaaggtg gacaatgccc tgcagtctgg gaactctcaa 480

gagagtgtga cagagcagga cagtaaagac agcacctata gcctcagcag caccctgacc 540

ctgtctaaag ccgactatga aaaacacaaa gtgtatgcct gcgaagtgac ccatcagggg 600

ctcagctctc ccgttaccaa gagctttaac cgaggcgaat gt 642

<210>36

<211>214

<212>PRT

<213>Synthetic

<400>36

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg

20 25 30

Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ser Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ser Gly Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>37

<211>1329

<212>DNA

<213>Synthetic

<400>37

gaagtgcagc tggtggagtc cggaggcgga ctggtgaagc ctggaggctc cctgaggctg 60

tcctgtgccg cttccggctt caccttctcc gacgactaca tggcctggtt caggcaggcc 120

cctggaaaga ggctggagtg ggtggcttcc atctcccacg gcggcgacta catctactac 180

gccgacaacc tgaagggcag gttcaccatc tccagggaca acgccaagaa ctccctgtac 240

ctgcagatga actccctgag ggccgaggac accgccgtgt acttctgctc cagggacagg 300

aggtccatcg actattgggg ccagggcacc ctggtgacag tgtcctccgc aagtaccaag 360

ggacctagtg ttttccctct tgcaccttgc tccaggtcaa catcagagtc cacagctgct 420

cttggatgtc tcgttaagga ctacttccca gagccagtta ccgtatcctg gaactccgga 480

gctttgacaa gcggcgttca tacattccca gctgtgttgc agagttctgg gttgtacagt 540

ttgagctcag tggtgaccgt gccttcatct tctttgggca ctaagaccta cacctgcaac 600

gtggatcaca agccaagcaa caccaaggtg gataagaggg tggagtccaa gtacggccca 660

ccatgtcctc catgtccagc ccctgaattt ttgggcgggc cttctgtctt tctgtttcct 720

cctaaaccta aagataccct gatgatcagc cgcacacccg aagtcacttg tgtggtcgtg 780

gatgtgtctc aggaagatcc cgaagtgcag tttaactggt atgtcgatgg cgtggaagtg 840

cataatgcca aaactaagcc ccgcgaagaa cagttcaaca gcacttatcg ggtcgtgtct 900

gtgctcacag tcctccatca ggattggctg aatgggaaag aatataagtg caaggtgagc 960

aataagggcc tccccagcag catcgagaag actattagca aagccaaagg gcagccacgg 1020

gaaccccagg tgtacactct gcccccctct caggaggaga tgactaaaaa tcaggtctct 1080

ctgacttgtc tggtgaaagg gttttatccc agcgacattg ccgtggagtg ggagtctaat 1140

ggccagcccg agaataatta taagacaact ccccccgtcc tggactctga cggcagcttt 1200

ttcctgtatt ctcggctgac agtggacaaa agtcgctggc aggagggcaa tgtctttagt 1260

tgcagtgtca tgcatgaggc cctgcacaat cactatacac agaaaagcct gtctctgagt 1320

ctgggcaaa 1329

<210>38

<211>443

<212>PRT

<213>Synthetic

<400>38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asp

20 25 30

Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser His Gly Gly Asp Tyr Ile Tyr Tyr Ala Asp Asn Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ser Arg Asp Arg Arg Ser Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn LysGly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>39

<211>642

<212>DNA

<213>Synthetic

<400>39

gacatccaga tgacccagtc cccttcctcc ctgtccgctt ccgtgggcga cagggtgacc 60

atcacctgca gggccagcca ggacatctcc aactacctgt cctggtacca gcagaagccc 120

ggcaagacca tcaagctgct gatctactac acctccaggc tgcacagcgg cgtgcctagc 180

aggttctccg gttctggctc cggcaccgac tacaccttca ccatcagctc cctgcagccc 240

gaggacatcg ccacctactt ctgccagcag ggcaagaccc tgccctacac cttcggccag 300

ggcaccaagg tggagatcaa gcgcactgtg gctgccccca gtgttttcat atttcccccc 360

agtgatgagc aactgaagtc cggcacagcc tctgttgtat gtctgctgaa taatttttat 420

ccacgggagg ccaaggtgca gtggaaggtg gacaatgccc tgcagtctgg gaactctcaa 480

gagagtgtga cagagcagga cagtaaagac agcacctata gcctcagcag caccctgacc 540

ctgtctaaag ccgactatga aaaacacaaa gtgtatgcct gcgaagtgac ccatcagggg 600

ctcagctctc ccgttaccaa gagctttaac cgaggcgaat gt 642

<210>40

<211>214

<212>PRT

<213>Synthetic

<400>40

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Thr Ile Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>41

<211>422

<212>PRT

<213>Homo

<400>41

Val Pro Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys

1 5 10 15

Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly

20 25 30

Val Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro

35 40 45

Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp

50 55 60

Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly

65 70 75 80

Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu

85 90 95

Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg

100 105 110

Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg

115 120 125

Ala Leu Ser Cys Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr

130 135 140

Ala Ser Pro Pro Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn

145 150 155 160

Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg

165 170 175

Asn Arg Gly Gln Gly Arg Val Pro Val Arg Glu Ser Pro His His His

180 185 190

Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser

195 200 205

Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser

210 215 220

Ile Met Tyr Asn Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu

225 230 235 240

Thr Val Tyr Ala Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu

245 250 255

Pro Ala Gly Val Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro

260 265 270

Pro Gly Gly Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe

275 280 285

Thr Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr

290 295300

Cys His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu

305 310 315 320

Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu

325 330 335

Gly Lys Leu Leu Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe

340 345 350

Val Trp Ser Ser Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro

355 360 365

Trp Leu Glu Ala Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys

370 375 380

Gln Leu Tyr Gln Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr

385 390 395 400

Glu Leu Ser Ser Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala

405 410 415

Leu Pro Ala Gly His Leu

420

<210>42

<211>17

<212>PRT

<213>Synthetic

<400>42

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 510 15

Ala

<210>43

<211>9

<212>PRT

<213>Synthetic

<400>43

Leu Gln Ser Gly Ser Ser Pro Pro Thr

1 5

<210>44

<211>10

<212>PRT

<213>Synthetic

<400>44

Trp Gly Gln Gly Thr Lys Val Glu Ile Lys

1 5 10

<210>45

<211>10

<212>PRT

<213>Synthetic

<400>45

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1 5 10

<210>46

<211>30

<212>PRT

<213>Homo

<400>46

Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu Ala Pro Gly Pro His

1 5 10 15

Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro Arg Arg Tyr

20 25 30

<210>47

<211>9

<212>PRT

<213>Homo

<400>47

Pro His Pro Ala Ala Pro Ser Ser Trp

1 5

<210>48

<211>12

<212>PRT

<213>Homo

<400>48

Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu Ala

1 5 10

<210>49

<211>19

<212>PRT

<213>Homo

<400>49

Ala Ala Ala Pro Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala

1 5 10 15

Pro Ser Ser

<210>50

<211>9

<212>PRT

<213>Homo

<400>50

Ala Ala Ala Pro Gly His Pro Leu Ala

1 5

<210>51

<211>232

<212>PRT

<213>Homo

<400>51

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser LeuThr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210>52

<211>233

<212>PRT

<213>Staphylococcus

<400>52

Ser Glu Lys Ser Glu Glu Ile Asn Glu Lys Asp Leu Arg Lys Lys Ser

1 5 10 15

Glu Leu Gln Gly Thr Ala Leu Gly Asn Leu Lys Gln Ile Tyr Tyr Tyr

20 25 30

Asn Glu Lys Ala Lys Thr Glu Asn Lys Glu Ser His Asp Gln Phe Leu

35 40 45

GlnHis Thr Ile Leu Phe Lys Gly Phe Phe Thr Asp His Ser Trp Tyr

50 55 60

Asn Asp Leu Leu Val Asp Phe Asp Ser Lys Asp Ile Val Asp Lys Tyr

65 70 75 80

Lys Gly Lys Lys Val Asp Leu Tyr Gly Ala Tyr Tyr Gly Tyr Gln Cys

85 90 95

Ala Gly Gly Thr Pro Asn Lys Thr Ala Cys Met Tyr Gly Gly Val Thr

100 105 110

Leu His Asp Asn Asn Arg Leu Thr Glu Glu Lys Lys Val Pro Ile Asn

115 120 125

Leu Trp Leu Asp Gly Lys Gln Asn Thr Val Pro Leu Glu Thr Val Lys

130 135 140

Thr Asn Lys Lys Asn Val Thr Val Gln Glu Leu Asp Leu Gln Ala Arg

145 150 155 160

Arg Tyr Leu Gln Glu Lys Tyr Asn Leu Tyr Asn Ser Asp Val Phe Asp

165 170 175

Gly Lys Val Gln Arg Gly Leu Ile Val Phe His Thr Ser Thr Glu Pro

180 185 190

Ser Val Asn Tyr Asp Leu Phe Gly Ala Gln Gly Gln Tyr Ser Asn Thr

195 200 205

Leu Leu Arg IleTyr Arg Asp Asn Lys Thr Ile Asn Ser Glu Asn Met

210 215 220

His Ile Asp Ile Tyr Leu Tyr Thr Ser

225 230

Claims (25)

1. An antibody or antigen-binding fragment thereof that binds human LAG-3, characterized in that it binds to an epitope of human LAG-3 comprising the amino acid sequence: AAAPGHPLA (SEQ ID NO: 50).

2. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 1, which binds an epitope of human LAG-3 comprising the amino acid sequence: GPPAAAPGHPLA (SEQ ID NO: 48) or AAAPGHPLAPGPHPAAPSS (SEQ ID NO: 49).

3. An antibody or antigen-binding fragment thereof that binds human LAG-3, comprising:

(a) heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 11 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 13, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 14 or SEQ ID NO: 43 is shown; or

(b) Heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 15, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 16, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 17 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 18, and the amino acid sequence of the L-CDR2 is shown as SEQ ID NO: 19, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: shown at 20.

4. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-3, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

5. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 4, wherein the antibody is a monoclonal antibody.

6. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-3, wherein the antibody is a murine, chimeric, or humanized antibody.

7. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 6, wherein the antibody is a humanized antibody.

8. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-3, wherein the antigen-binding fragment comprises a Fab fragment, a F (ab') 2 fragment, a Fv fragment.

9. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

(a) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 4 is shown in the specification;

(b) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 6, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 8 is shown in the specification;

(c) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 22, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 24; and

(d) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 26, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 28.

10. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 9, wherein the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 heavy chain constant regions and a light chain constant region selected from the group consisting of kappa or lambda light chain constant regions.

11. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 10, wherein the heavy chain constant region is an IgG4 heavy chain constant region and the light chain constant region is a kappa light chain constant region.

12. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 11, wherein the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID NO: 30, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO: shown at 34.

13. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 9-12, wherein the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain and a light chain selected from the group consisting of:

(a) the amino acid sequence of the heavy chain is shown as SEQ ID NO: 32, and the amino acid sequence of the light chain is shown as SEQ ID NO: 36 is shown; and

(b) the amino acid sequence of the heavy chain is shown as SEQ ID NO: 38, and the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 40.

14. A nucleotide sequence encoding the antibody or antigen-binding fragment thereof that binds human LAG-3 according to any one of claims 1-13.

15. The nucleotide sequence of claim 14, wherein the nucleotide sequence comprises:

(a) as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 3, a nucleotide sequence encoding a light chain variable region;

(b) as shown in SEQ ID NO: 5, as shown in SEQ ID NO: 7, a nucleotide sequence encoding a light chain variable region;

(c) as shown in SEQ ID NO: 21, as shown in SEQ ID NO: 23, a nucleotide sequence encoding a light chain variable region; or

(d) As shown in SEQ ID NO: 25, as shown in SEQ ID NO: 27, and a nucleotide sequence encoding a light chain variable region.

16. The nucleotide sequence of claim 15, comprising the nucleotide sequence set forth as SEQ ID NO: 29, and the nucleotide sequence encoding the heavy chain constant region as set forth in SEQ ID NO: 33, and a nucleotide sequence encoding a light chain constant region.

17. The nucleotide sequence of any one of claims 14-16, comprising:

(a) as shown in SEQ ID NO: 31, as shown in SEQ ID NO: 35, a nucleotide sequence encoding a light chain; or

(b) As shown in SEQ ID NO: 37, as shown in SEQ ID NO: 39 encoding a light chain.

18. An expression vector comprising the nucleotide sequence of any one of claims 14 to 17.

19. A host cell comprising the expression vector of claim 18.

20. A method of producing an antibody or antigen-binding fragment thereof that binds human LAG-3 according to any one of claims 1-13, comprising the steps of:

(a) culturing the host cell of claim 19 under expression conditions such that the antibody or antigen-binding fragment thereof that binds human LAG-3 is expressed;

(b) isolating and purifying the antibody or antigen-binding fragment thereof that binds human LAG-3 of (a).

21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-13 and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition further comprises a PD-1 inhibitor.

23. The pharmaceutical composition of claim 22, wherein the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that binds PD-1.

24. Use of an antibody or antigen-binding fragment thereof that binds human LAG-3 according to any one of claims 1-13 or a pharmaceutical composition according to any one of claims 21-23 in the manufacture of a medicament for the treatment of cancer.

25. The use of claim 24, wherein the cancer comprises melanoma, renal cell carcinoma, non-small cell lung cancer, classical hodgkin's lymphoma, urothelial cancer, colorectal cancer, and liver cancer.

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